Project Summary/Abstract DNA synthesis has played a key role in the biotechnology revolution. The ready availability of synthetic DNA oligonucleotides and of genes assembled from them, has been invaluable for elucidating and unlocking biological function and enabling the new field of synthetic biology which can create novel cells, enzymes, therapeutics, diagnostics and other reagents of commercial value. Despite this impact, DNA synthesis uses chemical strategies developed over 30 years ago which are costly and limited to molecules of 200 nucleotides or less in length. Next-generation enzymatic DNA synthesis technologies are being explored that use template- independent DNA polymerases (TIDPs) for controlled addition of nucleotides to a growing DNA strand. Although advances have been reported recently, enzymatic DNA synthesis is still limited by the low efficiency of available TIDPs, and specifically by the relative inability of these polymerases to incorporate 3'-blocked nucleotides. In this Phase I Small Business Innovation Research (SBIR) project, Primordial Genetics Inc, a synthetic biology company with differentiated combinatorial genetic technology, and Denovium Inc., an artificial intelligence company pioneering novel Al methods for genetic discovery, are joining forces to develop novel and highly efficient TIDPs for enzymatic DNA synthesis in vitro. Denovium will use their computational capabilities based on machine learning algorithms to discover novel TIDPs with the desired activities from proprietary and public databases. Denovium will also perform proprietary artificial intelligence (AI) scans to determine the functional impact of all possible mutations on the selected TIDPs. Primordial Genetics will synthesize and express the resulting collection of sequences, and test them in vitro to identify the most active enzymes. The best 2 enzymes will be diversified using Primordial Genetics' proprietary Function Generator technology and other randomized diversification methods. Populations of genes encoding enzyme variants will be screened with ultra-high-throughput screens to identify the most active enzymes. The dataset resulting from this work will be used to train Denovium's sequence prediction algorithm to accelerate further enzyme improvements in Phase II. The proposed work is a feasibility study for isolating and developing novel enzymes suitable for enzymatic DNA synthesis, and also for creating a pipeline of enzyme optimization tools. The enzymes discovered and in this work will be directly useful for enzymatic DNA synthesis applications, and can be licensed or sold to leading DNA and gene manufaturers.